An industrially potent rhamnolipid-like biosurfactant produced from a novel oil-degrading bacterium, Bacillus velezensis S2

Surfactants can reduce the interfacial surface tension between two immiscible liquids making them a desirable component for various industrial applications. However, the toxic nature of chemical surfactants brought immense attention towards biosurfactants. Being biodegradable, biosurfactants are eco-friendly and considered safer for different commercial uses. This study focused on the production of biosurfactant from an oil-degrading bacteria and its functional efficacy for prospective industrial applications. Here, a promising oil-tolerant strain, Bacillus velezensis S2 was isolated from oil contaminated sites which showed >50% degradation of convoluted crude oil within 28 days in comparison to a control. The isolate was then found to produce an excellent surface-active compound with an emulsification index of 67.30 ± 0.8% and could reduce the surface tension up to 36.86 ± 0.36 mN m−1. It also manifested a critical micelle concentration of 45 mg L−1 while reducing the surface tension from 72 to 30 mN m−1. When extracting biosurfactant from isolated bacteria, ethyl acetate extraction showed 1.5 times greater efficacy than chloroform : methanol extraction. The purified biosurfactant was characterized using TLC, 1H NMR, 13C NMR, FTIR, elemental analyses and spectrophotometric techniques leading to its identification as a rhamnolipid. The stability of produced biosurfactant at higher temperature (up to 180 °C) was determined by thermal analysis, endorsing its application in high temperature reservoir conditions. Additionally, the extracted biosurfactant showed excellent foaming efficacy with insignificant antibacterial and cytotoxic responses, which indicates their potential application in cleaning and cosmetics industries. Thus, the present study outlines a bi-functional novel isolate Bacillus velezensis S2 which could play a significant role in oil remediation from the environment as well as serve as a potential source of non-toxic and eco-friendly biosurfactants for various industrial applications.


III. An additional extension at 72 °C for 5 min
The amplified products were analyzed by gel electrophoresis (PS300TP, Biometra, Germany) in a 1.5% agarose gel with a 100 bp DNA ladder (PROMEGA, USA) and visualized by using an UVP transilluminator (Analytik Jena, US).After conferring the amplification, PCR product was sequenced by an automated DNA sequencer (3500 genetic Analyzer, USA).Full length sequences (1400-1500 bp) were obtained using the Seqman Genome Assembler (DNAstar, USA) by combining the partial sequences, collected from using forward and reverse primers.
A phylogenetic tree was constructed using the neighbor-joining method showing the relationship of the isolate with other closely linked reference strains obtained from GenBank database of the National Center for biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/GenBank) using the basic local alignment search tool (BLAST).
The Kimura 2-parameter method was used to compute the numbers at nodes that indicate the percentages of 1000 bootstrap (Kimura 1980) in MEGA 6.0 software (Das and Tiwary 2013).
The sequence data of the isolates were deposited onto the GenBank nucleotide sequence database using the BankIt sequence submission under accession number OR337901.

Gravimetric Analysis of crude oil biodegradation
100 mL of n-hexane was taken in a 500 mL separating funnel containing 100 mL of cell free supernatant (CFS) with 10% crude oil and mixed vigorously.The separated organic layer was Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2024 eluted and filtered into a 250mL round flask bottle.The procedure was repeated thrice.After extraction, then n-hexane was evaporated in a water bath at 70-75ᵒC.The residue crude oil was then weighed by a digital balance (SHIMADZU, Japan).The quantitative degradation by gravimetric analysis was estimated using the following formula.
% degradation = X 100 Here, the weight of degraded crude oil was determined by subtracting the weight of recovered oil from culture media from the weight of the residue oil in the control media.

Spectrophotometric Method
The residual oil present in the culture media was extracted in n-hexane at an interval of seven days up to 28 days of culture incubation following the same procedure described in section 2.4.
The amount of extracted residual oil was then recorded by UV spectrophotometer (UV-2600, SHIMADZU, Japan) at wavelength of 337 nm using the standard curve for crude oil diluted in n-hexane.To calculate the degraded oil amount in percentage, following equation was used.The column consisted of SH Rtx-CLP, having a length of 30 mg, an inner diameter of 0.32 mm, and a film thickness of 0.50 µm.Here, the column temperature was started from 120 °C (Habibullah-Al-Mamun, Ahmed et al. 2019) (1 min hold) at a rate of 15°C/min to 200 °C (1 min hold) and then at a rate of 5°C/ min to 300 °C (10 min hold).The temperature of detector and injector was maintained at 300 °C and 200 °C, respectively.Nitrogen was used as the carrier gas with a flow rate of 10.0 mLmin −1 .The chromatograms were analyzed with GCsolution Postrun (Version 2.41.00) with a workstation (GCsolution), and a computer for data acquisition and processing.

Oil Spreading Assay (OSA)
40 mL of distilled water (Milli-Q) in a Petri plate with diameter of 20 cm was taken.Then 100 µL of crude oil was dropped onto the surface of the water, followed by the addition of 20 μL of CFS on the center of the thin oil layer.CFS was collected from an overnight incubated culture (1.2*10 8 CFU/mL) grown in MSM and 10% oil containing media for 24 h and 37•C.After the placement of CFS, if the oil is displaced and a clear zone is formed on the oil-water interface, the diameter of the clear zone was measured and compared to 20 µL of distilled water as a negative control (Mouafi, Elsoud et al. 2016) and 20 µL of Tween 20 and SDS were used as positive controls.

Drop collapse test (DCT)
2 µl of crude oil was applied to 96-well microtiter plates.5µl of CFS was added to the surface of the oil in the well followed by the equilibration of the plate for 1 h at 37°C.The drop shape was being observed for 1 min using a magnifying glass.The result was considered positive for biosurfactant production when the drop collapsed and if the drops remained intact or round, they scored as negative indicating the lack of biosurfactant production.In this experiment, distilled water was used as negative control treatment, while Tween 20 and SDS were used as positive control (Korayem, Abdelhafez et al. 2015).

Emulsification Index (E 24 ):
The test was performed by mixing two milliliters of crude oil (2 mL) with 2 mL of CFS in a test tube and vortexing vigorously at high speed for 2 min.The mixtures were left to stand for 24 h, and the emulsion index (E 24 ) was calculated as indicated in the following formula: E24 (%) = total height of the emulsified layer/total height of the liquid layer × 100.
Here, Tween 80 and PBS were used as positive and negative controls respectively.

Bacterial adhesion to hydrocarbon (BATH) assay
The cells were first washed couple of times to remove any interrupting solutes and then resuspended in a buffer salts solution (pH 7.0) containing 16.9 g of K 2 HPO 4 , 7.3 g of KH 2 PO 4 , 0.2 g of MgSO 4 .7H 2 0, and 1.8 g of urea per liter to give an optical density (OD) of ~ 0.5 at 610 nm.Following that, 1 mL of crude oil was added to the 4 mL of cell suspension in a screwcap test tube (18ml, 16x150mm) and the test tube was vortex-shaken for 3 min.After shaking, aqueous phases and crude oil were allowed to separate for 1 hr.After careful removal of aqueous phase with a Pasteur pipette, the OD of the aqueous phase was then measured at 610 nm.The hydrophobicity of cells is expressed as the percentage of their adherence to crude oil, which is calculated as follows: % of bacterial cell adherence = 1-( x 100 ℎ  ℎ   ℎ   )

Surface Tension Determination
The CFSs were collected from five different conc. of grown culture (1.3*10 8 , 3.2*10 8 , 8.0*10 8 , 2*10 9 and 2*10 3 ) with OD value of 0.2, 0.4, 0.6, 0.8 and 0.98 respectively.The syringe was blunt shaped and had a needle of 0.47 mm diameter.Prior to measurement, the glass slides were cleaned thoroughly with deionized water, dried, and then placed.Each droplet excreted from syringe was 5 µL with each measurement repeating three times.Here Deionized (DI) water and sterile nutrient broth was used as a control (Nitschke, Ferraz et al. 2004).
-            ) ) X 1002.4.3Oil Degradation Analysis by Gas Chromatography (GC)100 mL of n-hexane was taken in a 500 mL separating funnel containing 100 mL of CFS with 10% crude oil and mixed vigorously.The separated organic layer was eluted and filtered into a 250mL round flask bottle.The procedure was repeated thrice.After extraction, hexane was then evaporated up to 2 mL in a water bath at 70-75 •C(Varjani, Rana et al. 2015).The extract containing residue crude oil in n-hexane was then further filtered by a micro-syringe filter (ECOSTAR, HANGHZOU) and collected in GC sample vial for instrumental analysis.The biodegradation of crude oil was examined by injection a 1.0 µL aliquot of final extract into a GC-ECD (Shimadzu 2010 plus) equipped with a 63 Ni electron capture detector and a moving needle-type injection system, where the injection mode was splitless.